The Tumor Necrosis Factor/Nerve Growth Factor (TNF/NGF) receptor superfamily represents a growing family with over 20 members identified so far in mammalian cells. Although the receptors of this superfamily differ in the primary sequence of their extracellular domains, the TNF/NGF receptor superfamily members share cysteine rich subdomains that are thought to adopt generally similar tertiary folds. (Bazan, 1993; Beutler and van Huffel, 1994; Smith et al., 1994). Except for two receptors, the p55 TNF receptor and Fas/APO1, the various members of this receptor family may have varying structural differences. Nevertheless, there is much similarity of function between the receptors, indicating that they share common signaling pathways. One example for this similarity is the ability of several receptors of the TNF/NGF family to activate the transcription factor NF-κB (see hereinbelow).
TRAF2 is a member of a recently described family of proteins designated TRAF (TNF Receptor Associated Factor) that includes several proteins identified as, for example, TRAF1, TRAF2 (Rothe, M., et al (1994); PCT published application WO 95/33051), TRAF3 (Cheng, G. et al. (1995), TRAF4 (CART1, C-rich motif associated with RING and TRAF domains, Regnier et al. 1995), TRAF5 (Ishida et al. 1996a, Nakano et al. 1996) and TRAF6 (see Cao et al. 1996a, Ishida et al. 1996b). All proteins belonging to the TRAF family share a high degree of amino acid identity in their C-terminal domains, while their N-terminal domains may be unrelated. As shown in a schematic illustration of TRAF2 (FIG. 1 herein), the molecule contains a ring finger motif and two TFIIIA-like zinc finger motifs at its N-terminal end. The C-terminal half of the molecule includes a region known as the “TRAF domain” containing a potential leucine zipper region extending between amino acids 264-358 (called N-TRAF). An additional domain towards the carboxy end of the molecule between amino acids 359-501 (called C-TRAF) is responsible for TRAF binding to the receptors and to other TRAF molecules to form homo- or heterodimers.
Recruitment of TRAF adapter proteins to the cytoplasmic domains of receptor molecules can lead to the assembly of larger signaling complexes that consist of distinct TRAF adapter molecules and other effector proteins with enzymatic functions. Numerous reports have examined the activation of intracellular kinases in response to TRAF-dependent signal transduction. In particular, kinases of the mitogen-activated protein kinase (MAPK) family have been shown to be key players for signaling pathways that are triggered by TRAF-containing complexes. These pathways appear to culminate in c-Jun amino-(N)-terminal kinase (JNK) activation (Reinhard et al. 1997; Song et al. 1997). TRAF proteins can thus serve to modulate the ability of receptors to trigger distinct signaling pathways that lead to phosphorylation and activation of protein kinases and, subsequently, to the activation of transcription factors of the Rel and AP-1 family.
The c-Jun transcription factor is phosphorylated at its amino terminus by JNK, the most downstream member of one MAPK signaling pathway (Hibi et al. 1993). To be activated JNK needs to be phosphorylated by a MAPK kinase (MAPKK, SEK, MEK).
This kinase itself is phosphorylated by a MAPKKK (MEKK1), which can be activated through phosphorylation by GCKR (germinal center kinase related) protein, the most upstream kinase described in this pathway (Minden et al. 1994; Lin et al. 1995; Shi and Kehrl 1997). Dominant-negative mutants of either of these proteins that lack kinase activity block TRAF-mediated JNK activation that is induced by members of the TNF/NGFR superfamily. Thus, TRAF proteins appear to regulate the JNK activation pathway at a very proximal step (Liu et al. 1996; Lee et al. 1997; Reinhard et al. 1997). Cells from TRAF2-deficient mice failed to activate JNK in response to TNFα (Yeh et al. 1997). JNK has been demonstrated to mediate the integration of a co-stimulatory signal by CD28 during activation of T lymphocytes (Su et al. 1994). Taken together, these results suggest that co-stimulation by CD28 and TRAF-mediated co-stimulation, after ligation of TNFR-related molecules, utilize the same distal signaling components.
TRAF proteins also appear to play an important role in modulating an early step in receptor-induced activation of NF-κB (Rothe et al. 1995b; Cao et al. 1996; Nakano et al. 1996). NF-κB comprises members of a family of dimer-forming proteins with homology to the Rel oncogene which, in their dimeric form, act as transcription factors. These factors are ubiquitous and participate in regulation of the expression of multiple genes. Although initially identified as a factor that is constitutively present in B cells at the stage of Igκ light chain expression, NF-κB is known primarily for its action as an inducible transcriptional activator. In most known cases NF-κB behaves as a primary factor, namely the induction of its activity is by activation of pre-existing molecules present in the cell in their inactive form, rather than its de-novo synthesis which in turn relies on inducible transcription factors that turn-on the NF-κB gene. The effects of NF-κB are highly pleiotropic. Most of these numerous effects share the common features of being quickly induced in response to an extracellular stimulus. The majority of the NF-κB-activating agents are inducers of immune defense, including components of viruses and bacteria, cytokines that regulate immune response, UV light and others. Accordingly, many of the genes regulated by NF-κB contribute to immune defense (see Blank et al., 1992; Grilli et al., 1993; Baeuerle and Henkel, 1994, for reviews).
One major feature of NF-κB-regulation is that this factor can be found in a cytoplasmic non-DNA binding form which can be induced to translocate to the nucleus, bind DNA and activate transcription. This dual form of the NF-κB proteins is regulated by I-κB—a family of proteins that contain repeats of a domain that was initially identified in the erythrocyte protein ankyrin (Gilmore and Morin, 1993). In the unstimulated form, the NF-κB dimer occurs in association with an I-κB molecule which imposes its cytoplasmic localization preventing its interaction with the NF-κB-binding DNA sequence, and activation of transcription. The dissociation of I-κB from the NF-κB dimer constitutes its critical activation step by many of its inducing agents (DiDonato et al., 1995). There is so far little understanding of the way in which cell specificity is determined in terms of responsiveness to the various NF-κB-inducing agents.
Evidence that TRAF proteins can influence receptor-mediated activation of NF-κB came from the demonstration that dominant-negative forms of TRAF2 can inhibit NF-κB activation in response to oligomerization of several TNFR-related molecules, including TNFRII, CD40, CD30, 4-1BB, and Ox40 (Rothe et al. 1994, 1995b; Duckett et al. 1997; Arch and Thompson 1998). However, gene elimination studies in mice have failed to implicate a required role for a specific TRAF in NF-κB activation by any of these receptors (Lee et al. 1997; Yeh et al. 1997). This suggests that receptor engagement may activate NF-κB by more than one pathway.
One of the most potent inducing agents of NF-κB is the cytokine tumor necrosis factor (TNF). There are two different TNF receptors: the p55 and p75 receptors. Their expression levels vary independently among different cells (Vandenabeele et al., 1995). The p75 receptor responds preferentially to the cell-bound form of TNF (TNF is expressed both as a type II-transmembrane protein and as a soluble protein) while the p55 receptor responds just as effectively to soluble TNF molecules (Grell et al., 1995). The intracellular domains of the two receptors are structurally unrelated and bind different cytoplasmic proteins. Nevertheless, at least part of the effects of TNF, including the cytocidal effect of TNF and the induction of NF-κB, can be induced by both receptors. This feature is cell specific. The p55 receptor is capable of inducing a cytocidal effect or activation of NF-κB in all cells that exhibit such effects in response to TNF. The p75-R can have such effects only in some cells. Others, although expressing the p75-R at high levels, show induction of the effects only in response to stimulation of the p55-R (Vandenabeele et al., 1995). Apart from the TNF receptors, various other receptors of the TNF/NGF receptor family: CD30 (McDonald et al., 1995), CD40 (Berberich et al., 1994; Lalmanach-Girard et al., 1993), the lymphotoxin beta receptor and, in a few types of cells, Fas/APO1 (Rensing-Ehl et al., 1995) are also capable of inducing activation of NF-κB. The IL-1 type-I receptor, also effectively triggering NF-κB activation, shares most of the effects of the TNF receptors despite the fact that it has no structural similarity to them. Novel receptor subunits of the IL-18 receptor complex have been recently cloned and shown to trigger NF-κB translocation and activation in response to IL-18 (Born et al. 1998). The IL-1Rrp as well as a novel protein of the IL-1 receptor family, designated AcPL (Accessory Protein Like) are both required for IL-18 signaling.
The activation of NF-κB upon triggering of these various receptors results from induced phosphorylation of its associated I-κB molecules. Several components of a specific signal transduction cascade, activated in response to the proinflammatory cytokines TNF-α or IL-1β, have recently been identified. A novel protein kinase designated NIK for NF-κB Inducing Kinase was the first to be identified (see co-pending co-owned Patent Application WO 97/37016, Malinin et al. 1996). NIK was found to bind to TRAF2 and to stimulate NF-κB activation. NIK shares sequence similarity with MAP3K kinases and participates in the NF-κB inducing signaling cascade common to receptors of the TNF/NGF family and to the IL-1 type 1 receptor. TNF-α and IL-1β, initiate a signaling cascade leading to activation of two IκB kinases, IKK-1 [IKK-α] and IKK-2 [IKK-β], which phosphorylate IκB at specific N-terminal serine residues [S32 and S36 for IκBα S19 and S23 for IκBβ] (for review see Mercurio F and Manning A M, 1999). These kinases were identified as the components of a high molecular weight protein complex designated the IKK signalsome.
Phosphorylated IκB is selectively ubiquitinated by an E3 ubiquitin ligase, the terminal member of a cascade of ubiquitin conjugating enzymes. In the last step of this signaling cascade, phosphorylated and ubiquitinated IκB, which is still associated with NF-κB in the cytoplasm, is selectively degraded by the 26S proteasome. This process exposes the NLS, therefore freeing NF-κB to interact with the nuclear import machinery and translocate to the nucleus, where it binds its target genes to initiate transcription.
The identification of several additional components of the IKK signalsome has given a clue to the potential mechanisms by which receptor activation might be linked to IKK activation. One of these is an NF-κB essential modulator designated NEMO. This murine protein was found to be essential for the activation of NF-κB in a flat cellular variant of HTLV-1 Tax transformed fibroblasts which is unresponsive to all tested NF-κB stimuli (Yamaoka et al. 1998). NEMO was shown to homodimerize and to directly interact with IKK2. The same protein was independently cloned by Kovalenko et al. (see co-pending co-owned Israel Patent Application Nos. 123758 and 126024) as a RIP-binding protein and designated RAP-2. NEMO was later independently cloned by two other groups as a non-kinase component of the IKK signalsome and designated IKKAP-1 (Mercurio F et al 1999b, Rothwarf D M et al 1998). The same protein was also cloned as an E3 interacting protein, which is an adenoviral protein, encoded by the early transcription region and functions to inhibit the cytolytic effects of TNF and was shown to interact with RIP kinase (Li Y et al 1998). These studies provide evidence that NEMO mediates an essential step of the NF-κB signal transduction pathway. Three receptor-associated proteins appear to take part in initiation of the phosphorylation cascade (see diagrammatic illustration in FIG. 2). TRAF2, which when expressed at high levels can by itself trigger NF-κB activation, binds to activated p75 TNF-R (Rothe et al., 1994), lymphotoxin beta receptor (Mosialos et al., 1995), CD40 (Rothe et al., 1995a) and CD-30 (unpublished data) and mediates the induction of NF-κB by them. TRAF2 does not bind to the p55 TNF receptor nor to Fas/APO1, however, it can bind to the p55 receptor-associated protein called TRADD and TRADD has the ability to bind to a Fas/APO1-associated protein called MORT1 (or FADD—see Boldin et al. 1995b and 1996). Another death domain containing serine/threonine kinase receptor-interacting protein, designated RIP (see Stanger et al., 1995) is also capable of interacting with TRAF2 as well as with FAS/APO1, TRADD, the p55 TNF receptor and MORT-1. Thus, while RIP was initially associated with cell cytotoxicity induction (cell death), its ability to interact with TRAF2 also implicates it in NF-κB activation.
TRAF molecules appear to be involved in the pathway leading to NF-κB activation. These associations apparently allow the p55 TNF receptor and Fas/APO1 to trigger NF-κB activation (Hsu et al., 1995; Boldin et al., 1995; Chinnaiyan et al., 1995; Varfolomeev et al., 1996; Hsu et al., 1996). The triggering of NF-κB activation by the IL-1 receptor occurs independently of TRAF2 and may involve a TRAF2 homologue—TRAF6 and a recently-cloned IL-1 receptor-associated protein-kinase called IRAK (Croston et al., 1995). TRAF6 and IRAK have been also shown to play an important role in IL-18-induced signaling and function (Kanarakaraj et al. 1999).
The signaling cascades that are initiated by receptor recruitment of either TRAF molecules or death domain containing adapter proteins are regulated by proteins that can interfere with specific steps by modifying the composition of the multiprotein complexes and/or by blocking protein-protein interactions and downstream effector functions. Several cytoplasmic molecules that bind to TRAFs have been identified. Among them A20, c-IAPs (cellular Inhibitors of Apoptosis), TRIP (TRAF interacting protein) and I-TRAF/TANK (TRAF interacting protein, TRAF family members-associated NF-κB activator). (Rothe et al., 1994; Rothe et al., 1995b; Cheng and Baltimore 1996; Lee et al. 1997; Roy et al. 1997) and two others, one of which is designated clone 9, which shows some sequence homology to the proteins of the present invention, and another designated clone 15 (see co-pending co-owned Patent Application WO 97/37016). Each of these proteins has been shown to be capable at least of binding, and some also of interacting with members of the TRAF family. Yet, the functional roles of these interactions have been demonstrated to be quite distinct. These proteins may be an important link in the ability of TRAF-dependent signal transduction to modulate cell survival. In fact it is not yet clear how TRAFs, trigger the phosphorylation of I-κB. There is also no information yet as to the mechanisms that dictate cell-specific pattern of activation of TRAFs by different receptors, such as observed for the induction of NF-κB by the two TNF receptors. The crystal structure of the TRAF domain of human TRAF has been recently solved (Park, Y. C. et al. 1999). The structure reveals a trimeric self-association of the TRAF domain, which provides an avidity-based explanation for the dependence of TRAF recruitment on the oligomerization of the receptors by their trimeric extracellular ligands.
Accordingly, as regards NF-κB activation and its importance in maintaining cell viability, the various intracellular pathways involved in this activation have heretofore not been clearly elucidated, for example, how the various TRAF proteins, are involved directly or indirectly.
Furthermore, as is now known regarding various members of the TNF/NGF receptor family and their associated intracellular signaling pathways inclusive of various adapter, mediator/modulator proteins (see brief reviews and references in, for example, co-pending co-owned Israel Patent Application Nos. 114615, 114986, 115319, 116588), TNF and the FAS/APO1 ligand, for example, can have both beneficial and deleterious effects on cells. TNF, for example, contributes to the defense of the organism against tumors and infectious agents and contributes to recovery from injury by inducing the killing of tumor cells and virus-infected cells, augmenting antibacterial activities of granulocytes, and thus in these cases the TNF-induced cell killing is desirable. However, excess TNF can be deleterious and as such TNF is known to play a major pathogenic role in a number of diseases such as septic shock, anorexia, rheumatic diseases, inflammation and graft-vs-host reactions. In such cases TNF-induced cell killing is not desirable. The FAS/APO1 ligand, for example, also has desirable and deleterious effects. This FAS/APO1 ligand induces via its receptor the killing of autoreactive T cells during maturation of T cells, i.e. the killing of T cells which recognize self-antigens, during their development and thereby preventing autoimmune diseases. Further, various malignant cells and HIV-infected cells carry the FAS/APO1 receptor on their surface and can thus be destroyed by activation of this receptor by its ligand or by antibodies specific thereto, and thereby activation of cell death (apoptosis) intracellular pathways mediated by this receptor. However, the FAS/APO1 receptor may mediate deleterious effects, for example, uncontrolled killing of tissue which is observed in certain diseases such as acute hepatitis that is accompanied by the destruction of liver cells.
In view of the above, i.e. that receptors of the TNF/NGF family can induce cell death pathways on the one hand and can induce cell survival pathways (via NF-κB induction) on the other hand, there apparently exists a fine balance, intracellularly between these two opposing pathways. For example, when it is desired to achieve maximal destruction of cancer cells or other infected or diseased cells, it would be desired to have TNF and/or the FAS/APO1 ligand inducing only the cell death pathway without inducing NF-κB. Conversely, when it is desired to protect cells such as in, for example, inflammation, graft-vs-host reactions, acute hepatitis, it would be desirable to block the cell killing induction of TNF and/or FAS/APO1 ligand and enhance, instead, their induction of NF-κB. Likewise, in certain pathological circumstances it would be desirable to block the intracellular signaling pathways mediated by the p75 TNF receptor and the IL-1 receptor, while in others it would be desirable to enhance these intracellular pathways.